The identification of bacteria can be carried out using biochemical, cultural, antibody recognition and molecular biological tests (Feng P C S and Hartman P A: Fluorogenic Assays for Immediate Confirmation of Escherichia coli. 1982. Falkow S, Habermehl K O. ed: Rapid Methods and Automation in Microbiology and Immunology. Springer-Verlag, Berlin 1985: 30–33. AOAC Official Methods of Analysis 1995. Pepper Ill., Gerba C P and Brendecke J W: Environmental Microbiology. A laboratory Manual. Academic Press 1995.)
Food and Water Hygiene
Biochemical Test and Culture Medium
The most probable number (MPN) is the common method for the detection and quantitation of E. coli in foods. This method detects E. coli on the basis of the bacteria's ability to ferment lactose with the evolution of gas. Other non-E. coli organisms also ferment lactose and, therefore, several selective enrichment steps are required in order to sequentially select for coliform bacteria and E. coli. 
This widely used MPN method has several limitations. Many clinical E. coli isolates are lactose negative and thus are not detected using the MPN method. The MPN method requires a minimum of about four days to determine the absence of E. coli in food products and about seven days are required to get confirmed results. The growth of some E. coli, including the serotype 0157:H7 strains, is severely inhibited by the selectivity of the EC broth at 45.5° C. and gas production in the MPN method is susceptible to interference by high levels of competitor organisms.
More rapid methods for detecting E. coli are needed because of the time and accuracy limitations of the MPN method. It has been reported that 94% to 97% of E. coli strains possess the B-D-glucuronidase that can be detected by specific hydrolysis of a synthetic substrate, 4-methylumbelliferyl-B-D-glucuronide (MUG), to a fluorescent end product. When MUG is incorporated into lauryl sulfate tryptose (LST) broth, 107 to 108 CFU/ml of E. coli will yield this fluorescent product which can be detected under longwave UV light. However, a number of enteropathogenic E. coli including serotype 0157:H7 strains, do not possess the B-D-glucuronidase enzyme, do not exhibit fluorescence in LST-MUG medium, and therefore yield false-negative results using the MUG method. In addition, the selectivity of the method is compromised by the fact that some Shigella, Citrobacter, Ecterobacter, Klebsiella, Salmonella, and Yersinia species also produce B-D-glucuronidase and therefore yield false-positive results.
Another widely used test, the Analytical profile index (API) test strips, produced by BioMerieux (France), may be used to obtain test results quickly. These consist of a series of miniature capsules on molded plastic strips, each of which contains a sterile dehydrated medium in powder form. Addition of water containing a bacterial suspension simultaneously re-hydrates and inoculates the medium. A rapid reaction is obtained because of the small volume of medium and the large inoculum used. The identification of the unknown bacterium is achieved by determining a seven digit profile index number and consulting the API profile recognition system. However, there are strains of E. coli that yield a low discrimination value with the API strips.
When this occurs, further identification with sugar test is required for affirmation. Acid production from sugars such as D-Adonitol, Cellobiose, Lactose and D-Xylose are additional biochemical test for differentiation of Escherichia species and related species.
DNA Probes
The use of genetic probes in the detection of microorganisms is popular because they obviate the need for pure cultures, and are specific, sensitive, fast and reliable (Fred C. Tenover: DNA probes for infectious diseases. CRC Press, Inc. 1989). In DNA probe test, it is essential to know something about the nucleotide sequence of the microorganisms under investigation.
Bacteria belonging to different families or strains can be differentiated on the basis of heterogeneity in genetic sequences. One approach is the identification and use of specific toxin genes of disease causing strains to distinguish them from the normal flora. Another approach makes use of the conserved and polymorphic sites that are found in bacterial 16S ribosomal RNA (rRNA) sequences not present in human 18 rRNA or human mitochondrial 12S rRNA. The combination of the polymerase chain reaction technique for gene amplification, followed by sequencing of polymorphic regions and phylogenetic analysis of the resulting sequence information can also assist in strain identification. (Relman et al. The New Engl J of Medicine, 327: 293–301, 1992, Kui et al. FEMS Microbiology Letters 57:19–24, 1989. DeLong et al. Science 243: 1360–1363, 1989).
The E. coli identification kit produced by gene-trak systems, Framingham, Mass., USA, uses DNA oligonucleotides that complement the 16S rRNA. This assay uses hybridization techniques to detect E. coli, non-coli Escherichia fergusonii and Shigella species.
Another way of identifying bacteria specific DNA probes is by using randomly cloned chromosomal fragments. This involves the cloning of restriction enzyme cleaved genomic DNA of a bacteria, and selection of specific clones by determining their hybridization profiles by hybridization against its own species-sequences and other species-sequences. Only clones that hybridize to sequences from the same species but the clones were derived from will be selected (Tenover FC: DNA Probes for Infectious Diseases. CRC Press 1989).
Gastrointestinal Infection
Colorectal cancer is one of the top three cancer killers in the world. Factors implicated in its etiology include inappropriate diet, environmental factors and lack of reliable diagnostic markers. Recently, greater understanding of the genetic predisposition to colon cancer has been achieved through the identification of genes responsible for such susceptibility (Cowell J K, ed: In Molecular Genetics of Cancer. Dunlop M G: Molecular genetics of colon cancer. 1995. 113–134). Despite intensive research efforts, the mortality rate from colorectal cancer has not declined dramatically over the last 40 years.
Markers associated with cancer initiation or progression are important in patient care. Tumours diagnosed at an early stage can usually be cured by surgical excision or polypectomy (surgical excision cures 90% of patients with adenoma or carcinomas that are confined to the mucosa). Patients with advanced disease have a poor prognosis as mortality increases to more than 90% after metastasis takes place.
The gastrointestinal tract is often exposed to a range of microorganisms. When bacteria come into contact with a susceptible host, they can establish either a transient presence, colonize the individual, infect the individual or evolve with the host. The outcome can either be harmless, acute illness or a chronic condition that may lead to a serious outcome (Gibson G R and Macfarlane G T: Human Colonic Bacteria: Role in Nutrition, Physiology, and Pathology. CRC Press, Inc., 1995).
Bacteria have been associated with inflammatory bowel disease such as ulcerative colitis and Crohn's disease (Giaffer et al. Gut 33:646–650, 1992, Cartun et al. Mod Pathol 6:212–219, 1993; Liu et al. Gastroenterology 108:1396–404, 1995). In addition, patients with pan-colitis of long duration are at risk of developing colorectal cancer (Wanebo H J: In Colorectal Cancer. Lev R: Precursors of Colon Carcinoma 1993; 158–163). Although frequently implicated, the role of bacteria in colon related disease remains ill-defined and controversial. The identification of bacteria in physical proximity to diseased tissue does not provide definitive proof of a causal relationship between a bacterium and the diseased condition. This is especially so when the bacteria are commonly found surrounding the tissue (Swidsinski et al. Gastroenterology 115:281–286, 1998), as is the case in the colon, and there is no additional information to differentiate between bacteria. It is perhaps more convincing if the bacterium can be shown to be positioned in-situ in the diseased tissue and when isolated and characterized found to possess properties that will substantiate its presence within the tissue.
The bacterium Helicobacter pylori is an accepted Group 1 (definite) biological carcinogen for gastric cancer and causes of related gastric conditions such as duodenal ulcer, gastric ulcer and ulcer complications. H. pylori attaches to and thrives on the gastric mucosa resulting in a chronic immunological response from the host. (Marshall, B. J. Gastroenterologist 1:241–247, 1993). It is not firmly established whether H. pylori has invasive properties. However, pathogenic strains have been identified that can cause epithelial cell damage and mucosal ulceration on an intragastric administration to mice (Telford et al. J Exp Med 179:1653–1658, 1994) The question remains whether H. pylori is the only important factor in the development of gastric cancer because of its high infection/disease ratio. The current consensus is that there may be other factors other than H. pylori infection that are also important in gastric cancer risk (National Institutes of Health Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease 1994). A separate study put forward the theory that a synergistic interaction between a non-invasive bacteria and other enteropathogens can facilitate invasion by the otherwise non-invasive bacteria (Geir Bukhowm and Georg Kapperud, Infection and Immunity 55:2816–2821, 1987).
Numerous in-vivo and in-vitro studies have vividly shown that microorganism carry transmissible tumorigenic genetic information. Mutagenesis in such instances is either by transposition or site-specific recombination facilitated by conjugation, transformation and transduction. This information is constantly being exploited scientifically in creating mutants (Sherratt D J (ed): Mobile genetic elements. Dale J W: Molecular genetics of bacteria. 2nd Edition. John Wiley and Sons Ltd. Oxford University Press 1995). In 1995, Couralin et al. showed that invasive strains of Shigella flexneri and E. coli can carry out gene transfer that are stably inherited and expressed by the mammalian cell progeny (Courralin et al., C. R. Acad. Sci. Paris 318:1207–1212, 1995). Therefore, it is quite possible that the persistent presence of bacterial genetic sequences in the nucleus of mammalian cells can lead to genetic instability that may ultimately give rise to a tumour cell.
Bacterial invasion can stimulate similar a pattern of protein phosphorylation to that induced by growth factor (e.g. EGF) and cellular proliferative responses may then be altered with consequences for disease progression. (Galan et al. Nature 357:588–589, 1992). In addition, bacterial disruption of cell-cell interaction may affect cell proliferation patterns and differentiation (Epenetos A A and Pignatelli M (ed): Cell Adhesion Molecules in Cancer and Inflammation; Pignatelli et al.: Adhesion molecules in neoplasia: An overview. Chapter 1:1–13. Harwood academic publishers 1995). Cytonecrotizing factors have been identified that can cause formation of large multinucleated cells and cells spreading in tissue cultures. (Denko et al. Experimental Cell Research 234:132–138, 1997; Lemichez et al., Molec Microbiol 24:1061–1070, 1997; Machesky, L. M. and Hall, A, TICB 6:304–310, 1996). Accordingly, the persistence presence of bacteria can cause cellular changes leading to cell disorientation, proliferation and changes in cell morphology.
One cancer causing effect of bacteria is when Agrobacterium tumefaciens, a soil phytopathogen, genetically transforms plant cells by the transfer of the tumour-inducing (Ti) plasmid to the plant genome where its integration and expression result in the crown gall phenotype. A crown gall is a tumorous proliferation of plant cells which are released from normal metabolic and reproductive controls (Hughes M A: Plant Molecular Genetics. Addison Wesley Longman Ltd. 1996).
People travelling across continents may suffer from traveler's diarrhoea as the bacteria they are exposed to are not common in their county. The assays/kits that are used for detecting microorganisms in the Asia-Pacific region are imported from other continents and these imported assays/kits may not be as sensitive or as specific for the bacteria in the Asia-Pacific region.
Microorganisms transmitted by water and food usually grow in the intestinal tract of man and animals and leave the body in the faeces. Bacteria are known to possess gene sequences that make them toxigenic, hemorrhagic, invasive and adherent to tissues. Acute bacterial infection is well documented but it is still not known that if bacteria that do not cause overt symptoms but persist and remain undetected in their host can cause diseases with time. Therefore, it is important that the assays that are available are sensitive and specific for a wide range of pathogens.
The E. coli genetic sequence is published. (Blattner et al. Science 277:1453–1474, 1997). Some of its genetic sequence has homology to other bacteria (Janda J M and Abbott S L: The Enterobacteria. Lippincott-Raven Press 1998). The inventor, in accordance with the present invention, has identified E. coli DNA sequences which are unique to the Escherichae family and furthermore has shown that biochemical and cultural tests presently available are not adequate for detecting this family of bacteria. The present polynucleotide sequence in the genome of strains of the Escherichieae genus (Escherichia and Shigella), have proven to be more informative than the agar plates EMB, MacConkey and MUG. They can be used to detect E. coli that is either EMB, MacConkey or MUG negative. The sequences are also found in 0157:H7 and 029:NM strains of E. coli. Therefore, the present molecular markers provide improved tools for the detection and characterization of E. coli. 
In addition, the invention permits the use of the sequence(s) to study the outcome of tissue infection in-situ. The present gene sequences are more specific than the gene-trak sequence (gene-trak systems) and the sequences can be amplified many-fold to increase their detection limit. This makes the present invention useful for studying the role of microorganism in gastrointestinal and other disease conditions. The presence of the polynucleotide sequence in cells can be located by the use of the polymerase-chain-reaction amplification technique in-situ followed by hybridization to the in-situ amplified signals with sequence specific DNA probe.
The identification of these specific polynucleotide sequence(s) that can be used to detect for the presence of strains of E. coli and Shigella and related microorganisms in food, water, fecal specimens, tissues, secretions and other biological, environmental and/or laboratory samples is important for health reasons as it enables one to check on the quality of food and water hygiene and monitor transmission of the microorganism. Sensitive detection techniques and methods for assessing the role of bacteria in clinical conditions will ultimately help in the control of harmful microorganisms.